COMMENTARY
COMMENTARY
Dynamics and clustering of IRE1α during ER stress
T. Kelly Rainbolta and Judith Frydmana,b,1
Cellular integrity is critically dependent on maintain-
ing protein homeostasis. Proteotoxic stresses chal-
lenging this balance elicit regulated protective
responses essential for organismal fitness. Approxi-
mately one-third of synthesized proteins are secreted
from cells and undergo biogenesis in the endoplasmic
reticulum (ER). Upon import into the ER, these proteins
are engaged by chaperone systems to facilitate either
folding or triage and removal through pathways of ER
quality control (1). The activity of ER quality control
is precisely regulated in response to protein folding
ER Stress
A lumen, connected through a transmembrane segment
IRE1 Activity
B Monomers
Dimers
Small Oligomers
Clusters
Large Oligomers Monomers
ER Lumen
Cytosol
Assembly Fusion Dissolution
Fig. 1. (A) In response to acute ER stress, IRE1 rapidly forms many small
oligomers (small green orbs) throughout the ER membrane. These oligomers
fuse and assemble into a few large clusters (large green orbs) during the first
hours of ER stress. Upon sustained ER stress, IRE1 clusters are eventually
resolved by dissolution back into the free IRE1 pool in the ER membrane. Cluster
assembly closely correlates with the induction of IRE1 activities such as
XBP1 splicing while dissolution occurs parallel to the shutoff of the IRE1 arm of
the UPR. (B) Depiction of the molecular events occurring during IRE1 cluster
formation. First, IRE1 is activated by assembly into dimers and small oligomers.
Then these oligomers fuse to form large clusters that promote IRE1 activity.
Finally, clusters are resolved by dissolution of IRE1 back into free monomers.
a
Department of Biology, Stanford University, Stanford, CA 94305; and bDepartment of Genetics, Stanford University, Stanford, CA 94305
Author contributions: T.K.R. and J.F. wrote the paper.
The authors declare no competing interest.
Published under the PNAS license.
See companion article on page 1533 in issue 3 of volume 117.
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1
To whom correspondence may be addressed. Email: jfrydman@stanford.edu.
First published February 4, 2020.
3352–3354 | PNAS | February 18, 2020 | vol. 117 | no. 7
stress through the unfolded protein response (UPR)
(2). The UPR relies on three transmembrane signaling
proteins, IRE1, PERK, and ATF6, each of which senses
protein misfolding in the ER through a luminal domain
and transmits to the cytoplasm and nucleus in order to
induce the expression of distinct subsets of ER chap-
erones and other factors that ameliorate ER stress (3).
In PNAS, Belyy et al. (4) employ advanced quantitative
imaging strategies to reveal the intricacies of the reg-
ulation of IRE1 during ER stress in the cellular context.
The most conserved of the three UPR sensors is
IRE1, which is present in organisms from yeast to
mammals. IRE1 is a transmembrane protein composed
of three distinct domains, a sensing domain in the ER
to a cytosolic regulatory kinase domain, and finally a
C-terminal RNase domain. Upon ER stress, IRE1 is acti-
vated to ultimately result in the noncanonical splicing of
the mRNA encoding the transcription factor XBP1. After
translation, the resulting XBP1-spliced transcription fac-
tor drives the expression of ER chaperones and lipid bio-
synthesis genes as a part of transcriptional program to
resolve protein misfolding in the ER (2). In higher eukary-
otes, IRE1 also down-regulates translation of ER-targeted
proteins through direct cleavage of ER-associated
mRNAs in a process known as RIDD (5). Evidence from
yeast and mammalian systems suggests two mecha-
nisms through which IRE1 senses increased levels of
protein misfolding in the ER. The luminal domain in-
cludes a segment that has been shown to bind directly
to the ER Hsp70 chaperone BiP, facilitated by its
cochaperone ERdj4 (6). The IRE1 luminal domain can
also bind directly to misfolded proteins in the ER
through an MHC-like peptide binding cleft (7). Mis-
folding sensed through these mechanisms leads to
IRE1 activation, which occurs through dimerization at
interfaces in both its ER luminal and cytosolic domains
that have been observed in crystal structures (8, 9). Be-
yond this, early microscopy studies in yeast and mamma-
lian cells with fluorescently tagged IRE1 demonstrated
the formation of large foci during ER stress, suggesting
the formation of higher-order oligomers of IRE1 (Fig. 1A)
www.pnas.org/cgi/doi/10.1073/pnas.1921799117
 (10, 11). While the formation of these IRE1 clusters is well docu-
mented, their nature has remained ambiguous since they are not
amenable to purification for biochemistry or structural studies.
What is the architecture of IRE1 clusters? Are they static or mo-
bile? How are they assembled and cleared?
Belyy et al. explore the dynamics of IRE1 oligomers in their cellular
context. To this end, they developed an IRE1α-mNeonGreen re-
porter leveraging the extreme brightness and improved photostability
of this next-generation florescent protein. High-resolution confocal
microscopy revealed that IRE1 clusters are asymmetric, prompting an
investigation at superresolution using structured illumination micros-
copy (SIM). SIM imaging of cells stressed with tunicamycin, an inhibitor
of N-linked glycosylation in the ER, revealed that IRE1 clusters have
complex and intricate architectures including structures forming
loops and coils that localize to branching points in the ER network.
Interestingly, colocalization with the general ER membrane marker
Sec61β was incomplete, suggesting that IRE1 clusters may exclude
other ER components and create an IRE1-rich microdomain. This
could be accomplished through local perturbation of the ER lipid
membrane, for instance by compression of the lipid bilayer in the
ER that has been attributed to the IRE1 oligomer (12). These
observations suggest the intriguing possibility that IRE1 creates
localized platforms that facilitate its function in sensing ER stress
and inducing the UPR.
Next, Belyy et al. investigated the dynamics of IRE1 clusters
across the timeline of acute ER stress. To this end, they developed
a clonal U2OS cell line expressing inducible tagged IRE1 and
leveraged automated imaging and analysis pipelines to move well
beyond prior microscopy studies of IRE1 clustering in scope and
depth. Live imaging of single-cell trajectories of IRE1 clustering
revealed significant heterogeneity in response to stress, despite
analyzing a clonal cell population. Notably, nearly one-half of
cells never formed detectable IRE1 clusters. Inherent variability
in stress responses could reflect differences in cellular states, such
as cell cycle progression or microenvironment, dictating how cells
experience stress; alternatively, they could reflect stochastic
variability in stress response transcription factor activity (13). The
observed latent diversity in how cells respond to stress suggests
the intriguing possibility that the UPR is not a monolithic response.
The extent and consequences of such diversity in cellular response
to ER stress will be of great interest to further explore.
Following only those cells that formed IRE1 clusters, Belyy
et al. investigated the dynamics and life cycle of these structures
upon prolonged ER stress. This analysis confirms prior observa-
tions that many small IRE1 clusters form initially, and then coalesce
into a few large clusters per cell, which ultimately dissipate during
sustained ER stress (Fig. 1B). The apparent fusion events occurring
between IRE1 clusters is reminiscent of the behavior of assemblies
undergoing liquid–liquid phase separation (14). In recent years,
numerous protein assemblies have been shown to form conden-
sates and coalesce through demixing by liquid–liquid phase sepa-
ration to regulate a wide variety of activities such as splicing and
signal transduction (15). Common features of phase-separated
structures are their dynamic exchange of monomers with bulk solu-
tion and their fluid droplet-like fusion behaviors. To reveal the na-
ture of IRE1 cluster dynamics, the authors performed fluorescence
recovery after photobleaching experiments, which surprisingly
demonstrated that, despite their fusion behavior, IRE1 clusters are
not phase-separated droplets since most IRE1 monomers in clusters
do not readily exchange with the free pool of IRE1 in the ER. In fact,
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only a small percentage of IRE1 in clusters is available for exchange,
suggesting that perhaps IRE1 forms a lattice-like structure where
Rainbolt and Frydman
exchange may only happen at the boundaries. This is reminis-
cent of the lattices formed by other signaling proteins such as the
bacterial chemotaxis system where stable assembly is thought to
promote receptor cooperativity and enhance system sensitivity
(16). Such structural cooperativity could contribute to sustaining
the splicing activity of IRE1 during ER stress or perhaps alter
RIDD substrate selection.
An interesting feature of IRE1 clusters revealed by the automated
imaging approach of Belyy et al. is their extensive motility. Analysis of
In PNAS, Belyy et al. employ advanced quantitative
imaging strategies to reveal the intricacies of
the regulation of IRE1 during ER stress in the
cellular context.
the trajectories of thousands of IRE1 clusters suggests their
motion is largely diffusive rather than the result of active
transport along the cytoskeleton. Perhaps most surprising is the
observation that IRE1 cluster diffusion is largely independent of
the size of clusters, suggesting their motion is constrained by other
cellular features.
One possibility is that IRE1 favors particular subdomains of the ER.
This is supported by previous observations that IRE1 accumulates
at mitochondria–ER contact sites, a domain of the ER referred to
as mitochondrial-associated membranes (MAMs) (17, 18). Enrich-
ment of IRE1 clusters to MAMs could facilitate signaling from mi-
tochondrial sources for integration during the ER stress response
in order to coordinate adaptation across organelles. Furthermore,
IRE1 has also been shown to scaffold filamin A and impact cyto-
skeleton dynamics through which clustering of IRE1 into discrete
ER domains could impact the fate of migrating cells (19). It will be
interesting to explore the interactions of clustered IRE1 with other
cellular structures through microscopy or proximity labeling ap-
proaches and further investigate how IRE1 clusters may influence
organelle cross talk during ER stress.
Sustained ER stress eventually leads to a shift in the UPR from a
prosurvival to procell death state (20). This shift is paralleled by a decline
in IRE1 activation and XBP1 splicing that correlates with a decrease in
clustered IRE1. This down-regulation of IRE1 clusters could either be
accomplished through selective targeting by degradation pathways or
dispersal of the clusters and return of IRE1 to the free monomer pool in
ER. In order to definitively test the mechanism of IRE1 cluster resolution,
the authors employed an optical pulse-chase approach using the pho-
toconvertible florescent protein mEos4b fused to IRE1. These photo-
conversion experiments further confirmed that IRE1 clusters are not
liquid-like condensates, since their largely immobile cores remain un-
mixed upon fusion. Imaging cluster dissolution upon continued stress
indicated that IRE1 clusters neither break up into smaller clusters
nor are they degraded. Instead, IRE1 molecules melt back into
the ER network in a sharp transition indicative of an external
regulatory signal (Fig. 1B). The dissolution cue remains to be
determined, but likely candidates are PERK-regulated dephos-
phorylation or the increase in free levels of chaperone ERdj4 on
the luminal face of the ER, which returns IRE1 to an inactive state
(6, 21). IRE1 cluster dissolution would terminate the transcriptional
and likely also RIDD activity of activated IRE1, leaving the cell to
return to homeostasis or, in the case of continues stress, to shift the
response to additional proapoptotic branches of the UPR.
Therapeutic targeting of IRE1 is gaining great interest to treat
a wide range of diseases, from cancers such as multiple myeloma
to metabolic disorders such as diabetes (22). However, as of yet,
PNAS | February 18, 2020 | vol. 117 | no. 7 | 3353
 small-molecule inhibitors or activators of IRE1 have not success-
fully transitioned to the clinic. The revealing characterization of
IRE1’s life cycle during ER stress presented in Belyy et al. dem-
onstrates the intricate complexity and dynamics regulating
IRE1 activity. Combined with the tools the authors have devel-
oped, this study provides a springboard to realize a more thorough
understanding of the function and regulation of IRE1, which could
aid in developing future therapeutic strategies targeting IRE1 to
ameliorate a variety of diseases.
Acknowledgments
We were supported by grants from the NIH (AG056290 and GM056433) and the
CHDI Foundation (to J.F.). T.K.R. was supported by a NIH National Research
Service Award (GM120947).

1 I. Braakman, N. J. Bulleid, Protein folding and modification in the mammalian endoplasmic reticulum. Annu. Rev. Biochem. 80, 71–99 (2011).
2 P. Walter, D. Ron, The unfolded protein response: From stress pathway to homeostatic regulation. Science 334, 1081–1086 (2011).
3 A. Korennykh, P. Walter, Structural basis of the unfolded protein response. Annu. Rev. Cell Dev. Biol. 28, 251–277 (2012).
4 V. Belyy, N.-H. Tran, P. Walter, Quantitative microscopy reveals dynamics and fate of clustered IRE1α. Proc. Natl. Acad. Sci. U.S.A. 117, 1533–1542 (2020).
5 M. Maurel, E. Chevet, J. Tavernier, S. Gerlo, Getting RIDD of RNA: IRE1 in cell fate regulation. Trends Biochem. Sci. 39, 245–254 (2014).
6 N. Amin-Wetzel et al., A J-protein co-chaperone recruits BiP to monomerize IRE1 and repress the unfolded protein response. Cell 171, 1625–1637.e13 (2017).
7 G. E. Karagöz et al., An unfolded protein-induced conformational switch activates mammalian IRE1. eLife 6, e30700 (2017).
8 A. V. Korennykh et al., The unfolded protein response signals through high-order assembly of Ire1. Nature 457, 687–693 (2009).
9 J. Zhou et al., The crystal structure of human IRE1 luminal domain reveals a conserved dimerization interface required for activation of the unfolded protein
response. Proc. Natl. Acad. Sci. U.S.A. 103, 14343–14348 (2006).
10 Y. Kimata et al., Two regulatory steps of ER-stress sensor Ire1 involving its cluster formation and interaction with unfolded proteins. J. Cell Biol. 179, 75–86 (2007).
11 H. Li, A. V. Korennykh, S. L. Behrman, P. Walter, Mammalian endoplasmic reticulum stress sensor IRE1 signals by dynamic clustering. Proc. Natl. Acad. Sci. U.S.A.
107, 16113–16118 (2010).
12 K. Halbleib et al., Activation of the unfolded protein response by lipid bilayer stress. Mol. Cell 67, 673–684.e8 (2017).
13 D. Popovic, B. Koch, M. Kueblbeck, J. Ellenberg, L. Pelkmans, Multivariate control of transcript to protein variability in single mammalian cells. Cell Syst. 7, 398–
411.e6 (2018).
14 D. Ricci et al., Clustering of IRE1α depends on sensing ER stress but not on its RNase activity. FASEB J. 33, 9811–9827 (2019).
15 S. Alberti, Phase separation in biology. Curr. Biol. 27, R1097–R1102 (2017).
16 A. H. Erbse, J. J. Falke, The core signaling proteins of bacterial chemotaxis assemble to form an ultrastable complex. Biochemistry 48, 6975–6987 (2009).
17 A. Carreras-Sureda et al., Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer
and bioenergetics. Nat. Cell Biol. 21, 755–767 (2019).
18 T. Mori, T. Hayashi, E. Hayashi, T. P. Su, Sigma-1 receptor chaperone at the ER-mitochondrion interface mediates the mitochondrion-ER-nucleus signaling for
cellular survival. PLoS One 8, e76941 (2013).
19 H. Urra et al., IRE1α governs cytoskeleton remodelling and cell migration through a direct interaction with filamin A. Nat. Cell Biol. 20, 942–953 (2018).
20 R. Jäger, M. J. Bertrand, A. M. Gorman, P. Vandenabeele, A. Samali, The unfolded protein response at the crossroads of cellular life and death during endoplasmic
reticulum stress. Biol. Cell 104, 259–270 (2012).
21 T. K. Chang et al., Coordination between two branches of the unfolded protein response determines apoptotic cell fate. Mol. Cell 71, 629–636.e5 (2018).
22 C. Hetz, E. Chevet, H. P. Harding, Targeting the unfolded protein response in disease. Nat. Rev. Drug Discov. 12, 703–719 (2013).
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